The mortality from cancer mostly arises from late diagnosis at which time current therapeutics are ineffective. Although proteomics using mass spectrometry and other techniques enable characterization of proteins in serum, plasma, and urine, there is still a lack of useful early markers for the vast majority of cancer types (Rai et al., Ann. NY Acad. Sci. (2004) 1022:286-294; Diamandis, J. Natl. Cancer Inst. (2004) 96:353-356).
Historically candidate tumor markers were identified using monoclonal antibodies against tumor cell extracts (Fidler, Cancer Research (1978) 38:2651-2660). Screening and evaluation of these candidates has been the traditional method of identifying novel tumor markers. However, this technology has had limitations. It is labor intensive and time consuming to evaluate large numbers of candidate markers.
It has also been very difficult to identify markers that are sensitive and specific for a particular type of cancer. It is still difficult to identify markers for diagnosis, prognosis, staging, recurrence, and detection of minimal residual disease for most types of cancer.
SELDI-TOF mass spectrometry technology that is currently used for serum analysis is not capable of detecting any serum component at concentrations of less than 1 μg/mL (Lai et al., Proc. Natl. Acad. Sci. USA (2002) 99:3651:3656). This range of concentrations is approximately 1000-fold higher than the concentrations of known tumor markers in the circulation (Table 1) (Lai, supra):
TABLE 1Approx.Proteinconcentration,Classical tumor markerspmol/LCancer typeAlpha-fetoprotein150Hepatoma, testicularProstate-specific antigen140ProstateCarcinoembryonic antigen30Colon, lung, breastHuman choriogonadotropin20Testicular,choriocarcinomaHuman choriogonadotropin-β2Testicular,subunitchoriocarcinomaReference: Diamandis, supra.
Gene-trap vectors mark endogenous genes and enable the detection of changes in gene expression. Marking a gene enables the study of a specific promoter and the function of the corresponding gene. However, gene-trap vectors, most of which are plasmid or retrovirus-based vectors, have been limited by low efficiency, short-term expression or restriction to dividing cells. Recently developed HIV-1-based lentiviral vectors have overcome these obstacles and are increasingly being used for gene delivery in vitro. These vectors have resulted in long term gene expression in vivo in cells of the central nervous system (CNS), hematopoietic system, retina, muscle, liver, and pancreatic islets (Lai, supra).
HIV-1 lentiviral vectors integrate into dividing and nondividing cell genomes and stably express the transgene. Two HIV-1-based lentiviral vector derivatives, pZR-1 and pZR-2, have been developed for gene-trapping in mammalian cells in vitro and in vivo (Lai, supra). These lentiviral gene-trap vectors contain a reporter gene, either β-lactamase or green fluorescent protein (GFP), that is inserted into the U3 region of the 3′ long terminal repeat. Both of the trap vectors readily integrate into the host genome by using a convenient infection technique and result in GFP or β-lactamase expression. This technique facilitates rapid enrichment and cloning of the trapped cells. The reporter gene is driven by an upstream, cell-specific promoter (Lai, supra).